Post-translational modifications (PTMs) is one of the major events causing the appearance of neo cryptic epitopes able to activate the immune system. Citrullination and carbamylation are well known examples of PTMs that generate targets of autoantibodies. Enzymatic modification of arginine in citrulline in histones is the prerequisite of NETosis, it is conceivable that other post translational modifications may occur during this event. PTMs of proteins may elicit an autoimmune response that leads to autoantibodies formation. Several autoimmune diseases seem to share these pathogenic mechanisms, in particular Anti-Phospholipid Syndrome (APS), an autoimmune entity defined by clinical and laboratory criteria, associated with thrombotic and/or obstetric morbidities. Some patients with clinical profile suggestive of APS are persistently negative for the routinely used antiphospholipid antibodies (aPL), these cases are known as "Seronegative APS" (SN-APS).
Since, dysregulation in PTMs of specific proteins represents triggers of processes potentially leading to autoimmunity, we will clarify how carbamylated- or acetylated-modified ß2GPI are able to activate the immune response and whether it may contain additional epitopes for anti-ß2GPI reactivity in those cases of SN-APS patients. We will explore whether these modified antigens are able to activate monocyte-derived immature DCs from healthy human donors.
Moreover, since both NETosis and extracellular microvesicles (MV) seem to be active in several autoimmune conditions, contributing to the formation of inflammation-induced thrombosis associated with autoimmune diseases, it will be interesting to characterize NET components and extracellular MV in the circulation of APS and SN-APS patients, correlating the new findings with clinical data and the presence of anti-Car-ß2GPI antibodies. This creates a link between innate immunity and autoimmunity that may explain autoantibodies formation.
The presence of autoantibodies is one of the hallmarks of autoimmune diseases. Several recent data show different autoantibody systems recognizing post-translationally modified proteins. In this regard, it is known that there is a range of possible PTMs of mammalian proteins that can allow immune recognition of neo-self epitopes. In fact, some PTMs can modulate the conformation, function, activity and/or location of a protein creating new self-antigens by altering immunologic processing and presentation. In this context our work is certainly very innovative, because ß2GPI represents the main target antigen in the Antiphospholipid Antibody Syndrome (APS) and, in relation to the heterogeneous clinical manifestations of autoimmune diseases, PTMs may be proposed as candidate processes that trigger autoimmune pathogenesis. Our research group has already studied some PTMs of ß2GPI, in particular, we have demonstrated that glucose-modified ß2GPI (G-ß2GPI) is a target antigen of humoral immune response in patients with APS and anti-G-ß2GPI titre was associated significantly with venous thrombosis and seizure.
This study can be considered a very up to date topic finding to clear "enigma" of autoimmune diseases, where until now the molecular mimicry was the prevailing hypothesis explaining generation of autoimmunity, thus, PTMs could arise as an additive factor which is required to start an autoimmune response. The knowledge of new pathogenic mechanisms, including the molecular pathways involving ß2GPI, might identify novel therapeutic targets and therefore improve the management of patients with APS in which antibodies against different antigenic targets may be detected. New technologies that allow us to identify "new" epitopes, may represent useful tools for a better efficiency and accuracy in the diagnosis of autoimmune diseases. In particular, the new diagnostic approaches should point out the risk stratification of the disease. Moreover, it will allow us to review new treatment strategies for APS that may target different pathways of coagulation and immunomodulation. In this vein, PTMs of proteins constitute the basic step of NETosis. NET composition is enriched by many proteins that are potentially post-translationally modified. Modified proteins are considered immunogenic and represent potent stimuli for formation of autoantibodies. Since NETosis seems active in several autoimmune conditions that are characterized by different auto-antibody profiles it is likely that different NET proteins and posttranslational modifications are active during NETosis. A strong effort must be done to characterize all NET components in different autoimmune diseases by proteomics and tentatively correlating the new findings with clinical data. A second issue is to quantify the kinetic of NET formation in different autoimmune conditions. Furthermore, studies have shown that microvesicles may carry DAMPs, eg, oxidatively modified membrane lipids, HMGB1, DNA and posttraslational modified proteins. Given their contribution to sterile inflammation and autoimmune disease, it is important to identify those molecules carried by microvesicles, and how DAMP-associated microvesicles contribute to autoimmune inflammation and disease. New technologic advances to isolate, quantitate, and characterize microvesicles are now available. Elucidating the molecular composition, pathogenic function, targeting, and uptake mechanisms of microvesicles is an important emerging research field in general and especially in autoimmune diseases. The signals extracellular vesicles transmit between cells also often function via modulating posttranslational modifications of target molecules, given that extracellular vesicles are carriers of several active enzymes catalysing posttranslational modifications. Posttranslational modifications of extracellular vesicles can also contribute to disease pathology by e.g. amplifying inflammation, generating neoepitopes or carrying neoepitopes themselves.
Final aim of this study is to determine whether the novel insights can be used for diagnosis or prognosis in those groups of patients persistently negative to conventional tests for detection of antiphospholipid antibodies (SN-APS). For SN-APS patients, to find "new" antigenic specificities of antiphospholipid antibodies could also be very useful to solve a social problem, because of the main clinical manifestations concerning the obstetric pathology. The identification of predictors of pregnancy outcome is necessary to streamline the design and use of new treatments acting on pathophysiological molecular targets, but unfortunately it is known that clinicians can not set a therapy without a clear diagnosis which requires at least one positive laboratory test in addition to clinical manifestations.